Deepest known novel species of the genus Ophiuroglypha Hertz, 1927 (Echinodermata: Ophiuroidea) from the central rift zone, Philippine Sea

. A new species of brittle star was collected in 2021 by the manned submersible “Fendouzhe” from the central rift zone deep waters, Philippine Sea, at a depth of 7729 m. It is described as Ophiuroglypha fendouzhe sp. nov., and is distinguished from its congeners based on the following features: slender arms, separated do rsal and ventral arm plates, overlapping large disc scales, and distally contiguous radial shields . We provide comprehensive descriptions of the external morphological features, including characteristics of the arm skeleton, and a phylogenetic analysis based on COI sequences. The interspeci ﬁ c genetic distance variation in the genus Ophiuroglypha found in this study was 3.89% to 24.25%. The new species constitutes the deepest known record for the genus Ophiuroglypha.


Introduction
The ophiuroid family Ophiopyrgidae Perrier, 1893 contains 154 valid species within 15 genera to date (Stö hr et al. 2022).In the present study, we focus on the genus Ophiuroglypha Hertz, 1927.Ophiuroglypha can be distinguished from other genera by usually having three small arm spines, and the middle spine becoming an upturned hooklet on distal arm segments (Hertz 1927;McKnight 2003).However, the Ophiuroglypha irrorata subspecies group includes species with up to nine arm spines, as well as straight middle and ventral spines at the distal end of the arm (Paterson 1985;McKnight 2003).These major European Journal of Taxonomy 891: 167-185 (2023) Segonzac 2005; Manso 2010; Olbers et al. 2019).The terminology for the oral papillae follows Hendler (2018).Additionally, we attempted to identify the species by molecular analysis (see below).

Morphological analysis
External morphological characters of the specimens were photographed through a dissecting stereo microscope (OLYMPUS SZX7).The arm skeleton was examined with a scanning electron microscope (SEM) Phenom ProX.For SEM examination, arm skeletal elements (ossicles) of the specimens were prepared by dissolving the soft tissue in undiluted NaOCl, washing the ossicles in water, then mounting them on SEM stubs and, after drying, coating them with gold.Holotype and paratypes are deposited at the Institute of Deep-sea Science and Engineering (CAS), Sanya, China.A tabular key to all species of Ophiuroglypha was compiled by synthesizing published descriptions.

DNA extraction, PCR amplifi cation and DNA sequencing
DNA extraction of the new species was done by using the TIANamp Marine Animals DNA kit (TianGen, Beijing) following the manufacturer's protocol.We sequenced the mitochondrial cytochrome c oxidase I (COI) partial gene for phylogenetic analysis.Primer sets COI005 (5´-TTAGGTTAAHWAAACCAVYTKCCTTCAAAG-3´) and COI008 (5´-CCDTANGMDATCATDGCRTACATCATTCC-3´) were used for amplifi cation of the COI partial gene (Hoareau & Boi ssin 2010;Okanishi & Fujita 2013).Total PCR mixture was 50 μL volume, containing 25 μL Premix Taq with 1.25 U Taq, 0.4 mM of each dNTP and 4 mMMg2+ (Ex Taq version, Takara, Dalian, China), 0.5 μM each of the primers and approximately 100 ng template DNA.The PCR temperature profi le for COI005/008 primer settings was as follows: an initial denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing temperature at 49ºC to 52ºC for 90 s, and extension at 72°C for 60 s; and a fi nal extension at 72°C for 10 min.COI partial gene sequences of the new species were deposited at NCBI GenBank (Table 1).

Phylogenetic analysis
We constructed a Maximum Likelihood (ML) phylogeny tree from COI, representing the family Ophiopyrgidae.For this, we used two sequences of the new species and additionally 15 sequences from GenBank.As outgroup we used COI sequences of Ophiacantha indica Ljungman, 1867 and Ophiosabine pentactis (Mortensen, 1936) (Table 1) from the family Ophiacanthida e. Ideally, as outgroup the nearest sister group, Ophiuridae, should be used, but due to the unresolved paraphyletic relationship between Ophiopyrgidae and Ophiuridae, this was not possible.All sequences were aligned using the Clustal W algorithm in MEGA X (Kimura 1980;Thompson et al. 1994;Kumar et al. 2018).The best-fi t substitution model of the partial COI gene in the ML tree was estimated by the "Find Best DNA/Protein Models" Option of MEGA X and the GTRGAMMAI (GTR+G+I) model (Kumar et al. 2018).The ML analysis was run by MEGA X, with a rapid bootstrap likelihood analysis, including 1000 bootstrap replicates.(Kumar et al. 2018).The genetic distances were analyzed according to the Kimura 2-parameter model (Kimura 1980) by using MEGA X (Kumar et al. 2018).The standard error of each group was discovered by performing 1000 bootstrap replications.

Results
The ophiuroid species found in the here studied collection could not be identifi ed by morphological or molecular data and we conclude that it is new to science.We describe it below as Ophiuroglypha fendouzhe sp.nov.A tabular key to all species of the genus Ophiuroglypha is provided in Table 3.

Molecular phylogenetic analysis
A 608 bp sequence of the COI gene was obtained after removing ambiguous aligned sites and successfully reconstructing the ML tree (Fig. 2) for 14 specimens from the genus Ophiuroglypha, and one specimen each from the genera Amphiophiura Matsumoto, 1915, Ophiura Lamarck, 1801 and Ophiogona Studer,    Tabular key to all species of the genus Ophiuroglypha Hertz, 1927, compiled from the literature.For convenience, the second oral tentacle pore is here labelled as the 1 st pore, because it is the fi rst visible pore along the arm, the actual fi rst pore being placed laterally in the oral plates (= jaw) and both ventral tentacle scales and adoral shield spines in second oral tentacle pore labelled as tentacle scales.Abbreviations: ASE = arm segment; DAP = dorsal arm plate; LOP = lateral oral papillae; TS = tentacle scale; VAP = ventral arm plate; VMT = ventralmost tooth.
European Journal of Taxonomy 891: 167-185 ( 2023) Tabular key to all species of the genus Ophiuroglypha Hertz, 1927, compiled from the literature.For convenience, the second oral tentacle pore is here labelled as the 1 st pore, because it is the fi rst visible pore along the arm, the actual fi rst pore being placed laterally in the oral plates (= jaw) and both ventral tentacle scales and adoral shield spines in second oral tentacle pore labelled as tentacle scales.Abbreviations: ASE = arm segment; DAP = dorsal arm plate; LOP = lateral oral papillae; TS = tentacle scale; VAP = ventral arm plate; VMT = ventralmost tooth.

Oral frame
Tentacle scales

References
Ophiuroglypha ossiculata (Koehler, 1908) up to 9, small, papilliform small, irregular more or less triangular, separated  (Lyman, 1878) and O. ambigua (Lyman, 1878).The closest relative of the new species is an unidentifi ed specimen that may represent a still undescribed species.Genetic distance values are provided in Table 2.

Diagnosis
Disc pentagonal, covered by large irregular overlapping scales.Radial shields broad, slightly wider than long, triangular, barely connected to each other at distal end, and proximal half separated by one elongated disc scale (Fig. 3A-C).Ventral disc covered by large, overlapping scales, slightly larger than dorsal disc (Fig. 3D).Jaw slender, large, contiguous, pair of pointed ventralmost teeth at apex, and up to fi ve lateral oral papillae (Fig. 3E).Second oral tentacle pore large, opening outside mouth slit with four adoral shield spines and fi ve ventral tentacle scales (Fig. 3E-F).Arms slender.Dorsal and ventral arm plates separated (Fig. 3F-H).Tentacle pores absent after seven or nine arm segments (Fig. 3I).

Etymology
T he specifi c name is dedicated to the manned submersible vessel 'Fendouzhe', which collected the specimen.

Description
MEASUREMENTS.Disc diameter 10 mm, arm base width 0.9 mm, and arm length 56 mm (Fig. 3).DISC.Disc pentagonal, covered by large irregular, and polygonal overlapping scales (Fig. 3A-C).Centrodorsal primary plate rounded in disc center (Fig. 3C).Radial shields broad, slightly wider than long, triangular, barely connected to each other at distal end, sharp pointed proximal edge, straight distal edge, and proximal half separated by one elongated disc scale (k-plate) (Fig. 3A, C-D).Median distal interradial area of disc formed by four to fi ve large scales (⅓ larger than other dorsal disc scales), most of them rounded rectangular (Fig. 3C).Ventral disc covered by large, overlapping scales, slightly larger than European Journal of Taxonomy 891: 167-185 (2023) on central dorsal disc (Fig. 3B, E-F).Distal end of genital slit bordered by small pointed genital papillae forming arm comb (Fig. 3E-F).Oral shield as wide as long, triangular, slightly convex at distal end, and connected to genital slit (Fig. 3E).Adoral shields small, narrow, rectangular, slightly curving outward from oral shield, contiguous proximally, and distally connected to fi rst lateral arm plate (Fig. 3E).Jaw slender, large, contiguous, with pair of pointed infradental papillae at apex, and spearhead-shaped teeth (Fig. 3E).Up to fi ve lateral oral papillae.Distalmost papilla (ventral compartment plate) much broader than others, and proximal two to three papillae spearhead-shaped to oval, and distal lateral oral papilla (buccal scale) less pointed and broader (Fig. 3E).Second oral tentacle pore large and opening outside mouth slit, with fi ve ventral tentacle scales and four adoral shield spines at each side.Genital slits conspicuous and extending from oral shield to disc periphery (Fig. 3E-F).
ARMS.Five slender, and non-moniliform arms.Dorsal arm plates small, diamond-shaped, straight to slightly fan-shaped with convex distal edge, pointed proximal edge, as wide as long, and widely separated, except on fi rst and second arm segments (Fig. 3D, G).First two ventral arm plates slightly wider than long, pentagonal, and contiguous.Following ventral arm plates wider than long, pentagonal to triangular, distally straight to slightly curved, proximally triangular, and increasingly separated along arm (Fig. 3E-F, H).Lateral arm plate with mostly three and rarely four arm spines.All arm spines small, smooth, pointed tip, similar in length, and approximately ½ arm segment long (Fig. 3G-L).Distally, middle arm spine slightly curved inwards (Fig. 3I-L).First tentacle pore with three or four tentacle scales (Fig. 3F-L).Second and third tentacle pores with four to two tentacle scales (Fig. 3F).Fourth to fi fth tentacle pores with three to one tentacle scales (Fig. 3F).Number of tentacle scales decreases to one scale, and absent after seven or nine arm segments (Fig. 3H, L).
COLOR.Creamy white in alcohol specimen (Figs 3, 5).OSSICLE MORPHOLOGY.(IDSSE-EEB-SW0251) Three arm spine articulations placed at distal edge of lateral arm plate, with clear muscle opening and small nerve opening (Fig. 4A-B).Tube foot pore developed as large within-plate perforation on lateral arm plates from base to middle part of arm, where tentacle scales begin to disappear from arm segments (Fig. 4A-B).Middle half of inner side of lateral arm plate with depression and two round knobs (Fig. 4C).Vertebrae with moderate zygospondylous articulation, proximal end with podial basins (Fig. 4D-H).Dorsal side of vertebrae distally triangular and proximally slightly curved, shallow longitudinal groove along midline (Fig. 4F-H).Ventral side of vertebrae with long and broad ambulacral groove with pair of lateral ambulacral canals (Fig. 4F-H).

Paratype variations
Four paratype specimens were collected from the same location.They are similar to the holotype (disc diameters 8.0-10.0mm), but one paratype has a sub-pentagonal disc and an obvious round centrodorsal primary plate (Fig. 5A).All paratypes are identical to the holotype with respect to the other morphological characters and this small variation is considered intraspecifi c (Fig. 5).

Distribution and habitat
So far only known from the type locality.

Discussion
The di agnostic characters of the new species agree with the description of the genus Ophiuroglypha, which is characterized by having at least three arm spines, the middle spine curved inwards (upturned hooklet) on distal arm segments, possession of genital papillae at genital slits, and a moderately sized oral shield (Hertz 1927;McKnight, 2003;Goharimanesh et al. 2021).However, there are some species of Ophiuroglypha without upturned hooklet (Lyman 1878;Lütken & Mortensen 1889;McKnight 2003;Olbers et al. 2019).Currently, Ophiuroglypha includes 18 species, and the new species Ophiuroglypha fendouzhe sp.nov.can be distinguished from its congeners by its sle nder arms, separated arm plates, large overlapping disc scales, and distally contiguous radial shields (Table 3).The present molecular study concurs with previous studies concluding that Ophiuroglypha is paraphyletic, and clusters with the genera Ophiogona Studer, 1876, Glaciacantha Fell, 1961, Spinophiura Stöhr & Segonzac, 2005, and Ophiura Lamarck, 1801(Christodoulou et al. 2019;O'Hara et al. 2019).Ophiogona differs from Ophiuroglypha by widely spaced disc scales with small granular coverage, fan-shaped ventral arm plate, and small ventral disc scales.Glaciacantha differs from Ophiuroglypha by marginal spinules or granules around the disc scales including the radial shields, a cluster of ventralmost teeth, and nine to ten long arm spines.Spinophiura differs from Ophiuroglypha by numerous spine-like lateral oral papillae and genital papillae, elongated tentacle scales, shape of oral shield, and six to seven elongated arm spines.

European Journal of
According to recent molecular studies some species of Ophiura may be closely related to Ophiuroglypha and also possess similar morphological features (Christodoulou et al. 2019).Ophiura fraterna (Lyman, 1878) differs from the new species by the number of arm spines (fi ve, minute, and unevenly spaced), contiguous dorsal arm plates, shape of oral shield (broader than long, pentagonal), and squarish papillae in the arm comb (Lyman 1878).Ophiura falcifera (Lyman, 1869) differs from the new species by numerous dorsal disc scales and their arrangement, separated pear-shaped radial shields (Lyman 1869(Lyman , 1883)).Ophiura tenera (Lyman, 1883) differs from the new species by irregularly rounded primary plates, contiguous radial shield, short and blunt arm spines, and contiguous dorsal arm plates (Lyman 1883).Hertz, 1927 is similar to Ophiuroglypha fendouzhe sp.nov. in number of arm spines, separate at proximal end but in contact at distal end of radial shields, and number of tentacle scales at the second tentacle pore, but differs in the middle arm spine being a glassy hook, disc scales small in the center but large distally, and contiguous ventral and dorsal arm plates (Hertz 1927).Ophiuroglypha ambigua (Lyman, 1878) is similar to O. fendouzhe sp.nov. in having separated ventral arm plates, and the number of tentacle scales at the second tentacle pore, but differs by the number of arm spines, separated radial shields, and contiguous dorsal arm plates (Lyman 1878).Ophiuroglypha arntzi (Manso, 2010) is similar to O. fendouzhe sp.nov. in the number of arm spines, the number of tentacle scales at second tentacle pore, and the separated ventral arm plates, but differs by separated radial shields, and contiguous dorsal arm plates (Manso 2010).Ophiuroglypha brevispinosa (H.L. Clark, 1915) is similar to O. fendouzhe sp.nov. in distally connected radial shields but differs by the shape of lateral oral papillae, number of arm spines, moderately large 5-6 primary plates, number of tentacle scales at second tentacle pore, and contiguous ventral and dorsal arm plates (Smith 1876;H.L. Clark 1915;Hertz 1927).Ophiuroglypha clemens (Koehler, 1904) differs from the new species by small arm spines (three and unevenly spaced), contiguous dorsal arm plates, and spaced dorsal disc scales (Koehler 1904).Ophiuroglypha carinifera (Koehler, 1901) is similar to O. fendouzhe sp.nov. in the number of arm spines, number of tentacle scales at second tentacle pore, and separated ventral arm plates, but differs by conical arm spines, separated radial shields, and contiguous dorsal arm plates (Koehler 1901).Ophiuroglypha costata (Lyman, 1878) is similar to O. fendouzhe sp.nov. in the number of arm spines, number of tentacle scales at second tentacle pore, and separated ventral arm plates, but differs by separated radial shields, peg-like arm spines, and contiguous dorsal arm plates (Lyman 1878(Lyman , 1882;;Bell 1905;Hertz 1927;Mortensen 1933;A.M. Clark 1952;Olbers et al. 2019).Ophiuroglypha euryplax (H.L. Clark, 1939) is similar to O. fendouzhe sp.nov. in number of arm spines, number of tentacle scales at second tentacle pore, and separated ventral and dorsal arm plates, but differs by separated radial shields, pentagonal centro-dorsal plate, and disc scales with tubercles (H.L. Clark 1939).Ophiuroglypha jejuna (Lyman, 1878) is similar to O. fendouzhe sp.nov. in the number of arm spines, number of tentacle scales at second tentacle pore, and separated ventral arm plates, but differs by tapering arm spines, smooth rounded disc scales, and contiguous dorsal arm plates (Lyman 1878).Ophiuroglypha kinbergi (Ljungman, 1866) is similar to O. fendouzhe sp.nov. in number of arm spines, number of tentacle scales at second tentacle pore, and separated ventral arm plates, but differs by arm spines being one arm segment in length, short pear-shaped and separated radial shields, and contiguous dorsal arm plates (Ljungman 1866).Ophiuroglypha lymani (Ljungman, 1871) is similar to O. fendouzhe sp.nov. in the number of arm spines, number of tentacle scales at second tentacle pore, and separated ventral arm plates, but differs by separated radial shields, separated rosette of fi ve rounded primary plates, and contiguous dorsal arm plates (Ljungman 1871).Ophiuroglypha ossiculata (Koehler, 1908) is similar to O. fendouzhe sp.nov. in the number of tentacle scales at the second tentacle pore, and separated ventral arm plates, but differs by the number of arm spines, separated radial shields, rounded primary plates, and contiguous dorsal arm plates (Koehler 1908).Ophiuroglypha plana (Lütken & Mortensen, 1899) is similar to O. fendouzhe sp.nov. in the number of arm spines, overlapping disc scales, and separated ventral arm plates, but differs by small and separated radial shields, number of tentacle scales at second tentacle pore, and contiguous dorsal arm plates (Lütken & Mortensen 1899).Ophiuroglypha rugosa (Lyman, 1878) is similar to O. fendouzhe sp.nov. in the number of arm spines, contiguous radial shields at the distal end, number of tentacle scales at second tentacle pore, and separated ventral arm plates, but differs by swollen scales with furrow, and contiguous wedge-shaped dorsal arm plates (Lyman 1878).Ophiuroglypha schmidtotti Hertz, 1927 is similar to O. fendouzhe sp.nov. in the number of arm spines, contiguous radial shields at the distal end, number of tentacle scales at second tentacle pore, and separated ventral arm plates, but differs by thick disc scales, broad lateral oral papillae, and contiguous dorsal arm plates (Hertz 1927).Ophiuroglypha scomba (Paterson, 1985) is similar to O. fendouzhe sp.nov. the in number of arm spines, contiguous radial shields at distal end, and separated ventral arm plates, but differs by small pear-shaped radial shields, distinct primary plate, number of tentacle scales at second tentacle pore, and contiguous dorsal arm plates on proximal half of the arm (Paterson 1985).Ophiuroglypha tumida (Mortensen, 1933) is similar to O. fendouzhe sp.nov. in the number of arm spines, and separated ventral and dorsal arm plates, but differs by slightly longer upper arm spine, separated radial shields, and number of tentacle scales at second tentacle pore (Mortensen 1933;Olbers et al. NETHUPUL H. et al., New species of Ophiuroglypha (Ophiuroidea) from the deep Philippine Sea 2019).Ophiuroglypha verrucosa (McKnight, 2003) is similar to O. fendouzhe sp.nov. in the number of arm spines, and separated ventral arm plates, but differs by separated radial shields, distinct disc scales, number of tentacle scales at second tentacle pore, and contiguous dorsal arm plates (McKnight 2003).
The molecular analysis of the genus Ophiuroglypha suggests that the new species is closely related to Ophiuroglypha rugosa and O. ambigua, but these two species differ from the new species by the number of arm spines, separated radial shields, contiguous dorsal arm plates, swollen scales with furrow, and contiguous wedge-shaped dorsal arm plates (Fig. 2, Tables 2-3).The interspecifi c genetic distance among species of Ophiuroglypha ranges from 3.89% to 24.25%, which agrees with B oissin et al. (2017).M ost species of Ophiuroglypha were found at a wider depth gradient than other ophiuroid species, and some of the species have been found from a shallow to abyssal (10-2000 m) depth range (Ophiuroglypha kinbergi and O. lymani) (OBIS 2022).Subspecies of Ophiuroglypha irrorata were recorded from a depth range of 403 m to 7340 m from all over the globe (Hertz 1927;Olbers et al. 2019).However, t he so far deepest species of Ophiuroglypha is Ophiuroglypha fendouzhe sp.nov.which was recorded at a depth of 7729 m.

Fig. 1 .
Fig. 1.Map of collecting station of the central rift zone, Philippine Sea, in this study.
asa = arm spine articulation asp = adoral shield spine cpp = central primary plate d = dorsal NETHUPUL H. et al., New species of Ophiuroglypha (Ophiuroidea) from the deep Philippine Sea dap

Fig. 2 .
Fig. 2. Maximum likelihood (ML) tree of the family Ophiopyrgidae, based on partial COI sequences (bootstrap support values were generated with rapid bootstrapping algorithm for 1000 replicates; red = new species).Abbreviation: C = Clade.

Table 2 .
Hertz, 1927roglyphaHertz, 1927, pairwise distance values based on 610 bp mitochondrial COI sequences, calculated using the Kimura 2-parameter method with 1000 bootstrap replicates (values in blue color represent standard error).

Table 3
Hertz, 1927on next two pages).Tabular key to all species of the genus OphiuroglyphaHertz, 1927, compiled from the literature.For convenience, the second oral tentacle pore is here labelled as the 1 st pore, because it is the fi rst visible pore along the arm, the actual fi rst pore being placed laterally in the oral plates (= jaw) and both ventral tentacle scales and adoral shield spines in second oral tentacle pore labelled as tentacle scales.Abbreviations: ASE = arm segment; DAP = dorsal arm plate; LOP = lateral oral papillae; TS = tentacle scale; VAP = ventral arm plate; VMT = ventralmost tooth.NETHUPUL H. et al., New species of Ophiuroglypha (Ophiuroidea) from the deep Philippine Sea